Loss of GSH and thiol enzymes in endothelial cells exposed to sublethal concentrations of hypochlorous acid

We investigated the effect of sublethal concentrations of hypochlorous acid (HOCl) on intracellular thiol groups. Exposure of human umbilical vein end othelial cells to HOCl caused a decrease in cell viability, with concentrat ions of less than or equal to 25 mu M HOCl being sublethal. At these concen trations, we saw a loss of glutathione and total protein thiol groups. Of t he thiol enzymes we investigated, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was particularly susceptible to inactivation, creatine kinase was m oderately susceptible, and lactate dehydrogenase was unaffected by HOCl at the concentrations used. Similar results were obtained with HOCl generated over 30 min by myeloperoxidase. GAPDH activity could be regenerated on rein cubation of cells in Hanks' balanced salt solution or reduction with dithio threitol. In contrast, glutathione loss was not reversible, and further dec reased with time. Cellular ATP levels decreased with sublethal HOCl concent rations and this appeared to be unrelated to the inactivation of GAPDH. Our results demonstrate that intracellular thiol groups differ in their reacti vity with HOCl and suggest that HOCl may be able to regulate specific cellu lar functions.